Metabolic Stability

Metabolic Stability

Metabolic stability - XenoGesisMetabolism is the predominant elimination mechanism for most drugs.

Although metabolism of drugs or xenobiotics in general, occurs in a variety of tissues such as the intestinal wall, lung, kidneys, skin and blood, the liver is regarded as the major site of drug metabolism. Hepatocytes or liver microsomes are, therefore, a fast and cost effective way to determine intrinsic clearance (CLint) of drug candidates.

These CLint determinations, along with other parameters including plasma protein binding and blood:plasma ratio, often allow a good prediction of in vivo hepatic clearance.

In order to mitigate against unacceptably high clearance it is usually necessary to identify the major metabolic pathway(s) as well as the nature of the enzymes involved.

At XenoGesis we know that every drug discovery project is different.

Whether a drug is designed to be administered orally, intravenously, or topically e.g. via inhalation will affect the type of In vitro assays needed. Pro-drugging strategies can avoid pre-systemic metabolism and help to deliver a drug to its target organ or site. All these approaches require specific In vitro experiments. XenoGesis can help you choose, plan and execute the studies that add the most scientific value to your drug discovery project.

XenoGesis offers a wide range of biological systems to determine in vitro metabolic stability including primary cells e.g. hepatocytes, tissue homogenates and tissue fractions e.g. microsomes, mitochondrial preparations, S9 fractions and intestinal fluid from a variety of preclinical species and human.

HEPATOCYTE METABOLIC STABILITY

Ian Dearman Media-99Hepatocytes, either fresh or cryopreserved, have been used very successfully to determine hepatic metabolism of drug research compounds. Hepatocyte suspensions are very powerful tools to study drug metabolism. On the one side they can be used to determine intrinsic clearance, which can then be easily scaled to predict In vivo clearance in the species of interest. On the other side hepatocyte suspensions can also be used to elucidate metabolic routes. In recent years major progress has been made in refining cryo-preservation techniques so that highly metabolically competent cells are available from all relevant preclinical species and man. Inter-individual variability, especially in human hepatocytes, has been overcome by the use of pooled cell preparations from as many as 100 individual donors. Hepatocytes are often referred to as the ‘gold standard’ In vitro system for an accurate estimation of the in vivo hepatic clearance mainly for two reasons.

Counted_HepsHepatocytes contain all phase I and phase II drug metabolising enzymes and their corresponding co-factors and for compounds which show poor passive permeability but are efficiently taken up by transport proteins, cell-free systems like microsomes may lead to significant over-predictions of In vivo metabolic clearance.

However, it should be mentioned that clearance via biliary excretion cannot be assessed by hepatocyte CLint assays.

 

The XenoGesis hepatocyte stability protocol

Format Cell suspension. 96 well with shaking
Hepatocytes Cryopreserved human; rat; dog; cynomolgus monkey; guinea pig; mouse hepatocytes (other species upon request)
Cell density 0.5 x 106/ml
Test compound concentration 1 µM
Number of time points 6
Incubation temperature 37°C
Analytical technique UPLC-MS/MS
Recommended number of replicates ≥ 2
Typical turnaround time 2 weeks

Note: Assay conditions can be tailored to client requirements.

To determine metabolic stability the test compound is mixed with a hepatocyte suspension. At set time points, small aliquots are withdrawn and the test compound concentration therein is measured by LC-MS/MS. The resulting concentration-time profile is then used to calculate intrinsic clearance (CLint) and half-life (t½).

The following example of dextromethorphan in a rat hepatocytes suspension shows how CLint and t½, are derived from the raw data.

gray_graph1

Metabolic stability of dextromethorphan in rat hepatocytes. Data are the means of triplicate determination ± one standard deviation.

For substrate concentrations significantly below Km metabolism follows first order kinetics, which means that the compound concentration-time profile can be described by:

gray_metabolic111(1)

 

Where C(t) is the compound concentration at time t, C(0) is the starting concentration and k is the elimination rate constant.

Natural logarithmic transformation (ln) allows us to fit a linear regression.

gray_graph2

ln(concentration) vs. time profile of dextromethorphan in rat hepatocyte suspension.

This ln(concentration) vs.time profile can be mathematically described by:

gray_metabolic211(2)

 

Where k represents the slope of the regression line.

Solving for k results in:

gray_microsomal311(3)

 

Once k is known, half-life (t½) can be calculated using:

gray_metabolic411(4)

 

And furthermore, CLint can be calculated by introducing the cell density ([cell]) in 106/ml:

gray_metabolic511(5)

MICROSOMAL METABOLIC STABILITY

Ian Dearman Media-207Liver microsomes are a well-established source of drug metabolising enzymes. They contain cytochrome P450 enzymes (CYP), flavin-containing monooxygenases (FMO) carboxyl esterases, epoxide hydrolase and UDP-glucuronosyltransferases (UGT). Liver microsomes are widely used to determine In vitro intrinsic clearance (CLint), which can then be scaled to predict In vivo hepatic clearance. Microsomes can also be used to elucidate metabolic routes. Liver microsomes are available from all relevant preclinical species and also from human. To overcome problems with inter-individual variability especially in humans, pooled liver microsomes from as many as 200 individual donors have become available.

To determine metabolic stability the test compound is mixed with liver microsomes. At set time points, small aliquots are withdrawn and the test compound concentration therein is measured by LC-MS/MS.

The XenoGesis microsomal stability protocol

Format 96 well with shaking
Microsomes Human; rat; dog; cynomolgus monkey; guinea pig; mouse microsomes (other species upon request)
Protein concentration 0.5 mg/ml
Test compound concentration 1 µM
Number of time points 6
Incubation temperature 37°C
Analytical technique UPLC-MS/MS
Recommended number of replicates ≥ 2
Typical turnaround time 2 weeks

Note: Assay conditions can be tailored to client requirements.

The following example shows how CLint and half-life (t½) are calculated from a concentration-time profile for dextromethorphan in rat liver microsomes. The resulting concentration vs. time profile is then used to calculate intrinsic clearance (CLint) and half-life (t½).

gray_graph3

Metabolic stability of dextromethorphan in rat liver microsomes. Data are the means of triplicate determination ± one standard deviation.

For substrate concentrations significantly below Km metabolism follows first order kinetics, which means that the compound concentration vs. time profile can be described by:

gray_microsomal111(1)

 

Where C(t) is the compound concentration at time t, C(0) is the starting concentration and k is the elimination rate constant.

Natural logarithmic transformation (ln) allows us to fit a linear regression.

gray_graph4

ln(concentration) vs. time profile of dextromethorphan in rat liver microsomes.

This ln(concentration) vs. time profile can be mathematically described by:

 

gray_microsomal211(2)

 

Where k represents the slope of the regression line. Solving for k results in:

 

gray_microsomal311(3)

 

Once k is known, half-life (t½) can be calculated using:

 

gray_microsomal411(4)

 

And furthermore, intrinsic clearance (CLint) can be calculated using equation 5, where [prot.] is the protein concentration in the incubation:

gray_microsomal511(5)

BLOOD OR PLASMA METABOLIC STABILITY

Certain drug metabolising enzymes (e.g. esterases, xanthine oxidase) are very active in extra-hepatic tissues, especially in blood or plasma. For orally administered drugs stability in plasma is usually mandatory to achieve sufficient exposure. For drugs that are administered via other routes, a rapid degradation in plasma can be an efficient way to minimise the risk of systemic side effects, whilst maintaining high exposure close to the site of administration. XenoGesis offers blood and plasma stability assays for a wide variety of preclinical species & strains and human.

S9 METABOLIC STABILITY

Ian Dearman Media-417The S9 fraction is a subcellular fraction that can be prepared from any tissue homogenate by centrifugation at 12000 x g. It is highly enriched in cytoplasm and microsomes. S9 tissue fraction contains a huge variety of phase I and phase II drug metabolising enzymes including CYPs, flavin-containing monooxygenases, esterases, epoxide hydrolases and UDP-glucuronosyltransferases (UGTs).

GASTROINTESTINAL METABOLIC STABILITY

Porcine small intestinal fluid (PSIF)

The small intestine is the major site of food digestion in the body. Shortly after its passage through the stomach, an orally administered (pro-)drug is exposed to a complex mixture of bile and pancreatic juice. Thus, the small intestinal contents is comprised of bile salts, phospholipids, cholesterol and bicarbonate as well as a cocktail of digestive enzymes including proteases, peptidases, lipases, carbohydrases and nucleases. The major proteases, trypsin and chymotrypsin are secreted as zymogens which require proteolytic activation in the small intestine.

Whilst fasted- or fed-state simulated gastric or intestinal fluids are powerful tools to investigate drug dissolution, their value to assess drug stability is limited, because they do not contain digestive enzymes. Porcine small intestinal fluid, on the other hand, is an excellent matrix to study pre-systemic stability of orally administered drugs or pro-drugs In vitro.

Metabolic Stability in Small Intestinal Mucosal Homogenate

The small intestinal mucosa is the innermost layer of the intestinal wall. It consists mainly of mucus and enterocytes. These cells contain a wealth of phase I and phase II enzymes, which can contribute significantly to low oral bioavailability. In the presence of relevant co-factors a homogenate prepared from the isolated small intestinal mucosa is a powerful and cost-effective tool to assess pre-systemic drug metabolism.